1. Field of the Invention
The present invention relates to an immobilized metal affinity chromatography (IMAC) instrument, a substrate containing immobilized metal affinity ligands, and a method for purifying and/or separating compounds containing a non-shielded purine or pyrimidine moiety or group using the instrument or substrate.
More particularly, the present invention relates to an immobilized metal affinity chromatography (IMAC) instrument and/or a substrate containing immobilized metal affinity ligands where the metal ion immobilized on the ligand of the column of an IMAC instrument or the substrate is capable of binding compounds containing a non-shielded purine or pyrimidine moiety or group where binding affinities results in a separation of different compounds containing a non-shielded purine or pyrimidine moiety or group or in a purification of compounds that do not contain a non-shielded purine or pyrimidine moiety or group from compounds that do contain a non-shielded purine or pyrimidine moiety or group. The present invention also relates to a method for purifying and/or separating compounds containing a non-shielded purine or pyrimidine moiety or group such as single stranded DNA, RNA, or other compounds containing a non-shielded purine or pyrimidine moiety or group, or to a method for removing compounds containing a non-shielded purine or pyrimidine moiety or group from compounds that do not contain a non-shielded purine or pyrimidine moiety or group such as removing nucleotides and primers from PCR reactions and/or reaction products and purifying plasmid DNA.
2. Description of the Related Art
Immobilized Metal Affinity Chromatography (IMAC) was introduced by Porath et al. (1, 2) as a means of purifying proteins based on the affinity of their surface-exposed amino acids (especially histidines) for chelated metal ions. The method has found widespread application in the purification of recombinant histidine-tagged and pharmaceutical proteins, most commonly using Cu(II) and Ni(II) ions chelated by iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) functionalities. Metal chelate ligands are best known as affinity agents in chromatography but have also been immobilized on foams (3), membranes (4), biosensor chips (5), and in electrophoresis gels (6); they have also been used as affinity precipitation agents (7).
The interaction of metal ions with nucleic acids is a long-standing and active field of study (8, 9). While metal ion binding to nucleic acids is well known, and plays an important role in the function of the widely used cancer drug cisplatin (10), IMAC has found very limited application in the purification of nucleic acids. Fanou-Ayi and Vijayalakshmi (11) and Hubert and Porath (12) demonstrated the binding of mononucleotides to copper IMAC resins, and histidine-conjugated PCR primers have been used to facilitate purification of the resulting histidine-tagged oligonucleotide products (13), but the potential applications of IMAC in nucleic acid separation and analysis remain largely unexplored.
Thus, there is a need in the art for an IMAC instrument and method, where the IMAC column includes metals or metal ions capable of binding compounds containing a non-shielded purine or pyrimidine moiety or group to effectuate separation and/or purification of different compounds containing a non-shielded purine or pyrimidine moiety or group or for purifying compounds that do not contain a non-shielded purine or pyrimidine moiety or group from compounds that do contain a non-shielded purine or pyrimidine moiety or group.